Culture medium and process for the biological production of alpha, omega-alkanedioic acid

ABSTRACT

An aqueous culture medium comprising (1) 3 to 35 grams per liter of alkali metal, calcium nitrate, magnesium nitrate, strontium nitrate and/or beryllium nitrate; (2) 0.3 to 10 grams per liter of alkali metal phosphate; (3) 1 to 15 grams per liter of an alkali metal acetate, calcium acetate, magnesium acetate, strontium acetate and/or beryllium acetate; and (4) a nutrient source; and a process for the production of an alpha,omegaalkanedioic acid of 10 to 16 carbon atoms comprising (1) introducing a mutated strain of Corynebacterium into a composition which has a pH of from 6 to 9 and which comprises 1 to 50 percent by volume of an n-alkane of 10 to 16 carbon atoms and a complemental amount of the culture medium above; (2) maintaining the composition at a temperature between 20* and 45*C. for 48 t0 96 hours; and (3) separating the alpha,omegaalkanedioic acid from the resulting mixture.

United States Patent [191 Dahlstrom et al.

[11.] 3,784,445 [451 Jan. 8, 1974 1 CULTURE MEDIUM AND PROCESS FOR THEBIOLOGICAL PRODUCTION OF ALPHA, OMEGA-ALKANEDIOIC ACID [75] Inventors:Robert Victor Dahlstrom; James Herbert Jaehnig, both of Manitowoc, Wis.

[73] Assignee: E. I. duPont de Nemours and Company, Wilmington, Del.

22 1 Filed: Dec. 30, 1971 21 Appl. No.: 214,379

[52] US. Cl 195/28 R, 195/30, 195/100 [51] Int. Cl Cl2b 1/00 [58] Fieldof Search 195/114, 3 H, 28 R,

[56] References Cited OTHER PUBLICATIONS A Compilation of Culture Mediafor Cultivation of Microorganisms, M. Levine & H. W. Schoenlein,Williams & Wilkins Co., 1930, pp. 31 & 32.

Primary Examiner-Lionel M. Shapiro Assistant Examiner-R. B. PenlandAttorney-William A. Hoffman [57] ABSTRACT An aqueous culture mediumcomprising (1) 3 to 35 grams per liter of alkali metal, calcium nitrate,magnesium nitrate, strontium nitrate and/or beryllium ni trate; (2) 0.3to 10 grams per liter of alkali metal phosphate; (3) l to 15 grams perliter of an alkali metal acetate, calcium acetate, magnesium acetate,strontium acetate and/or beryllium acetate; and (4) a nutrient source;and a process for the production of an alpha,omega-alkanedioic acid of10 to 16 carbon atoms comprising (1) introducing a mutated strain ofCorynebacterium into a composition which has a pH of from 6 to 9 andwhich comprises 1 to 50 percent volume of an n-alkane of rotate carbonatoms and a complemental amount of the culture medium above; (2)maintaining the composition at a temperature between 20 and 45C. for 48t0 96 hours; and (3) separating the alpha,omega-alkanedioic acid fromthe resulting mixture.

5 Claims, No Drawings CULTURE MEDIUM AND PROCESS FOR THE BIOLOGICALPRODUCTION OF ALPHA, OMEGA-ALKANEDIOIC ACID This invention relates tothe preparation of al pha,omega-alkanedioic acid from n-alkanes throughthe use of bacteria. Particularly, this invention relates to thepreparation of alpha,omega-alkanedioic acid of to 16 carbon atoms fromn-alkanes of 10 to 16 carbon atoms by the use of a mutant ofCorynebacterium. More particularly, this invention relates to a culturemedium for use in the preparation of alpha,omegaalkanedioic acid fromn-alkanes of 10 to 16 carbon atoms and a mutant of Corynebacterium andan improved process which utilizes the culture medium in the preparationof alpha,omega-alkanedioic acid of 10 to 16 carbon atoms.

lt was known that long chain alkanes could be oxidized utilizingCorynebacterium 7ElC (ATCC-l9067) to form their respective acids.However, only low yields and conversions were obtained in suchprocesses. Culture media, of course, were utilized but none of theseallowed for high yields and conversions of the alkanes to theirrespective alpha,omega-alkanedioic acid. Therefore, an improved culturemedium which would allow for such improved yields and conversions wassought. An improved process was also sought to further improve theyields and conversions of the alkanes to the alpha,omega-alkanedioicacid.

Such a culture medium has been found. It is aqueous and comprises 1) 3to 35 grams'per liter of a member selected from the class consisting ofalkali metal nitrate, calcium nitrate, magnesium nitrate, strontiumnitrate, beryllium nitrate and mixtures thereof; (2) a 0.3 to 10 gramsper liter of alkali metal phosphate; (3) l to grams per liter of analkali metal acetate, calcium acetate, magnesium acetate, strontiumacetate, beryllium acetate and mixtures thereof; and (4) a nutrientsource.

The improved process for the production of an alpha,omega-alkanedioicacid of 10 to 16 carbon atoms comprises (1) introducing a mutated strainof Corynebacterium capable of producing at high yield and conversionalpha,-omega-alkanedioic acid of 10 to 16 carbon atoms into acomposition which has a pH of from 6 to 9 and which comprises 1 to 50percent by volume of an n-alkane of 10 to 16 carbon atoms and acomplemental amount of the culture medium above; (2) maintaining thecomposition at a temperature of between about 20 to 45C. for 48 to 96hours; and (3) separating the alpha,omega-alkanedioic acid from theresulting mixture.

Some preferred mutated strains of the Corynebacterium are on file withthe American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Maryland 20852. These strains are referred to throughout bytheir ATCC number. The organism tentatively assigned to the genusCorynebacterium was first isolated by J. W. Foster (J. Bacteriology 85,859 1963). The original culture was pink but after a series of mutationswith chemical and physical mutagens the culture lost the capability ofproducing a pigment. The organism is a non-motile, gram rod withmetachromatic granules. Snapping division produces angular and palisadearrangement of cells. Older cells often lose gram characteristic withgranules still evident. The bacterium is an obligate aerobe and utilizesnitrates as sole source of nitrogen. The bacterium rods are 0.3 to 0.4by 0.7 to 0.8 micron. They grow well on most common laboratory media,but grow better on the culture medium of this invention. Colonies ofbacteria on tryptone glucose agar are dry, granular and small. Optimumtemperature of growth is 37C.

The amount of metal nitrate utilized in the culture medium depends onthe mutant of Corynebacterium which is used. A relatively large amountof nitrate is required for the bacterium utilizes it as the sole sourceof nitrogen. Preferably 6 to 30 grams per liter of the metal nitrate ispresent in the culture medium. The preferred nitrates are the alkalimetal nitrates, in particular sodium or potassium. The most preferredconcentration of the metal nitrate in the culture medium is 12 to 25grams per liter.

The alkali metal phosphate is used as a buffer compo nent in the culturemedium. A sufficient amount of the phosphate is present in the culturemedium to cause it to have a pH of from 6 to 9. Therefore, the amountutilized will vary according to the other components in the culturemedium. The preferred amount of alkali metal phosphate in the culturemedium is l to 6 grams per 1i ter. Preferred phosphates are K IHOP. andNaH PO H O.

The metal acetate is critical as a component in the culture medium inthat it significantly increases the yield of the alpha,omega-alkanedioicacid when the culture medium is used in the process of this invention.The preferred concentration for the metal acetate is from 2.5 to about10 grams per liter. Preferred acetates are the alkali metal acetates, inparticular, sodium and potassium.

The type of nutrient source in the culture medium is not critical.Examples of useful nutrient sources are tomato juice, broth solids,molasses plus yeast extract or cottonseed meal. The first two types ofnutrient source are preferred. The amount of nutrient source utilized isnormally from 1 to 5 grams per liter, preferably around about 2 gramsper liter of the culture medium.

The balance of the culture medium is made up of water. This can be tapwater or distilled water. Other components can be present in the culturemedium if they do not impede the function of the main components. Someof such components are MgSO '7H O, CaCl (neither of these two should beused in large amounts or they made cause the phosphate to precipitate),FeSO, with ethylenediamine tetraacetic acid chelating agent, MnSO -H O,H BO Na MoO -2H O, KI, and ZnSO '7H O. It is preferred that no ammoniumcompounds be present for they erode the ability of the me dium to causethe bacterium to accumulate acid. Copper compounds can be included inthe medium. However, they tend to give no advantage but instead resultin reduced acid production The culture medium can contain emulsifier.This is not required but does normally render the culture medium moreconducive to manipulation. Useful emulsifiers are substances such asEthofats (Armour and Company, Chicago, Illinois) which is monofatty orrosin acid esters of polyoxyethylene glycols having the general formulaExamples of other nutrient type additives which can be included in theculture medium are amino acids, vitamins, glucose, sugar, adipic acid,butyric acid and glutaric acid.

The culture medium which is discussed above is the medium which is usedin the process for preparing the alpha,omega-alkanedioic acid in thefull scale fermentation but is normally not used in the pregrowth of thebacteria. Pregrowth of the bacteria can be accomplished by placing alyophile culture of freeze-dried bacteria on a tomato juice agar slantwhich contains proteins and carbohydrates, i.e., tomato juice extractand glucose. The bacteria grows for from 24 to 48 hours at 37C. on theslant. A loopful of the bacteria is then transferred to a large flasksuch as a 125 ml. Erlenmyer for pregrowth. In the pregrowth stage thebacteria normally grows on a solution comprising 5 weight- /volumepercent molasses, preferably cane, 0.5 weight- /volume percent yeastextract, 0.15 weight/volume percent K HPO 0.06 weight/volume percentNail- P0," H and water. Weight/volume percent means grams/- millilitersin percent terms, i.e., 5 grams in 100 mls. is 5 weight/volume percent.

The pregrowth flask is maintained at about 37C. and vibrated at 220 rpmson a shaker with a 1 inch throw for 24 hours. The volume of the materialin the initial pregrowth flask is normally 25 ml. although this is notcritical. The pregrowth medium plus bacteria are built up by successivespiking of larger and larger volumes. When a pregrowth volume isobtained which is about percent of the total volume of materials to beused to make the alpha, omega-alkanedioic acid, the pregrowth stage iscompleted. At this point the pregrowth medium should contain at least 1X 10 organisms per ml. Normally, all the pregrowth medium is used toinoculate the culture medium and n-alkane.

The culture medium described above is useful in growing bacteria whichproduce acids from hydrocarbons. in particular, the culture medium isuseful in preparing alpha,omega-alkanedioic acids from n-alkanes of 10to 16 carbon atoms by using mutants of Corynebacterium. The processproduces higher levels of conversion and yield on the n-alkanes of 10 to14 carbon atoms. It is most useful on dodecane which producesl,l2-dodecanedioic acid. The n-alkane utilized can be one n-alkane or amixture of such. A mixture of acids then can be produced by the processof this invention.

In the recitation of the process above it is stated that theCorynebacterium mutant is capable of producing at high yields andconversions the alpha,omegaalkanedioic acid. When the process of thisinvention is utilized, this statement means to producealpha,omegaalkanedioic acid at yields and conversions so that thepercentage of the n-alkane converted to the alpha,omega-alkanedioic acidis at least 6.8 mole percent; preferably at least 10 mole percent. Thisis calculated by multiplying the yield times the conversion. Conversionis defined as the percentage amount in moles of the n-alkane convertedto other than the nalkane without consideration as to what it isconverted to while yield means the percentage amount in moles of then-alkane converted which is in the form of the alpha,omega-alkanedioicacid. Normally, when conversion is high, yield is lower and vice versa.Therefore, these can vary up and down but the product of the two isalways greater than the above recited percentages.

The composition inoculated by the bacteria in the process normallycomprises 1 to 50 percent by volume of the n-alkane with 5 to 20 percentby volume being preferred. The remainder of the composition is theculture medium as discussed above. Normally, the Corynebacterium mutantis added to the composition as part of an inoculum. This inoculumusually represents about 5 to 20 percent by volume of the compositionplus the inoculum.

The pH of the composition, i.e., the culture medium and n-alkane, shouldbe from 6 to 9 initially with the preferred range being from 7 to 8. Themost preferred range is from 7 to 7.5. The temperature of thecomposition should be from 20 to 45C. both at the time the bacterium isadded and during fermentation. The preferred range is 25 to 35C. Contactof the bacteria and the composition is for up to 96 hours. Higher yieldsand conversions are not obtained until about 48 hours and after 72 hoursdiminishing returns set in.

One method of separating the alpha,omegaalkanedioic acid from theresulting mixture is to (1) remove the cells and excess hydrocarbon fromthe aqueous phase by phase separation, (2) adjust the pH of the aqueouslayer to 2.0 with concentrated sulfuric acid, (3) filter out theprecipitate (alpha,omega-alkanedioic acid), and (4) wash the precipitatewith water. The purity of the acid is normally 50 to 65 percent byweight when this procedure is followed.

Mutants of the Corynebacterium which are useful in the process can beprepared by the standard techniques of (1) high energy radiation; (2)chemical mutation; (3) lethal synthesis and combinations thereof. Thetwo former techniques are preferred. The mutants referred to herein wereprepared by one or a combination of the three methods.

The culture medium and process are useful in preparing thealpha,omega-alkanedioic acid. The acids are useful as intermediates inthe preparation of polyamides which have low water absorption and goodstiffness and which can be formed into fibers.

The following Examples are meant to illustrate but not to limit theinvention. Parts and percentages are by weight unless otherwisespecified.

EXAMPLE I F reeze-dried mutant of Corynebacterium sp. G 1 l-25, ATCC21744 was grown on a tomato juice agar slant. A loopful of theCorynebacterium was transferred to 50 mls. of molasses-yeast broth in a300 ml. shake flask (Erlenmyer). The broth had been previouslyautoclaved for 15 minutes at 121C. The composition of the molasses-yeastbroth was as follows:

0.15 weight/volume percent K HPO.,,

0.06 weight/volume percent NaH PO -H O,

5 weight/volume percent cane molasses,

0.5 weight/volume percent yeast extract, and

remainder water. its pH had been adjusted to 7.0 to 7.1 with NaOH. Thebroth containing the Corynebacterium mutant was then incubated for 24hours at 37C. in a 1 inch throw rotary shaker at 220 rpms. 25 Mls. ofthis broth was then transferred to 250 mls. of fresh molasses-yeastbroth prepared as above in a 1 liter shaker flask. This broth wasincubated for 24 hours at 37C. in a 1 inch throw rotary shaker at 240rpms. This broth was then used as an inoculum for the main fermentationmedia test flasks.

The main fermentation test flasks were prepared as three days in arotary shaker at 220 rpms. with a l inch follows: throw.

1. A salts and buffer medium was prepared in dis- After the incubationperiod, all flasks were adjusted tilled water and contained thefollowing ingredients to the initial volume with distilled water. Ml.samin their respective concentrations: 5 ples were centrifuged forseparation of the residual hy- 0.04 weight/volume percent MgSO -7H 0drocarbon, cells, and aqueous fractions. The cells set- 0.3weight/volume percent K HOR, tled between the oil and aqueous phases.The 1,12- 0.12 weight/volume percent Nal-l POrH O dodecanedioic acidassay of the aqueous layer was 5 weight/volume percent NaNO done by thinlayer chromatography. Twenty-two milli- 0.003 weight/volume percent CaCl0 .moles of l,l2-dodecanedioic acid were produced per liter of thematerials in the fermentation test flasks at Ithe end of thefermentation. The percent (mole) yield Stock Solution Concentration .Tlf3. E l Pid i l.l.?.'?lfi9lll?l9lf 0.2 volume percent of stock solution iL FeSO. ethylene- EXAMPLE n di i h i H tetraacetic acid 0.125 grams/100mls. percent. Following the procedure of Example l, 1,12-

stock solution MnSO 'H,O so mgs/lOO mls. 2O dodecanedioic acid wasproduced from n-dodecane M i f: from a Corynebacterium sp. G 1 1-25,ATCC 21744.

0c 1. 3 13: 5 "figs/100 mg The amounts of sodium nitrate and sodiumacetate volume t of were varied in the culture medium as shown in TableSTOC SO [1011 Y I NZMMOOZHzO 43 mgslloo I. The results of the acidanalysis are also shown. When 0.2 volume percent of the nitrate wasbeing varied, the amount of sodium ace- 01 20 mes/100 .tate was 0.50Weight/volume percent while when the stock solution acetate was beingvaried, the amount of sodium nitrate Znsoflmo 80 "ms/100 was 2.50weight/volume percent.

TABLE I l,l2-Dodeca nedioic acid l: ConvermM/ Yield sion MEDIA VARIABLESliter (mole) (mole) l. laNaNO;=0.3l3% (weight/volume) 38 I8 72 2. %NaNO=0.625% (weight/volume) 38 I6 84 a. %NaN0,--1.25% (weight/volume) 1s 764. %NaNO,='-2.50% (weight/volume) 36 21 62 5. %NaC,H,-,O 0.O% (standard)I2 20 20 6. %NaC,H,0 =0.25% (weight/volume) 3| 22 7. %Nac,H,0,=0.50%(weight/volume) 34 25 54 8. %NaC,H,O,--l.00% (weight/volume) 20 2| 36 mMmillimoles The pH of the salts and buffer medium was adjusted. EXAMPLEIll] 9. 2.1. to .7 W. .N f M Following the procedure of Example l, 1,12-

dodecanedioic acid was prepared from dodecane using 6 Salts and buffermedium was dlluted with (115- the various Corynebacterium strains shownin Table ll.

tilled Water to 65 Percent of its Original Concentra' The weight/volumepercent of sodium nitrate utilized To the diluted Salts and buffermedium was was 2.5 rather than that of Example I. The results of theadded 0.3 weight/volume percent tomato juice and run are shown in TableIL 0.5 weight/volume percent sodium acetate. 20 Mls.

of this solution were added into a 300 ml. shaker TABLE H flask with 0.1ml. Ethofats (mono-fatty or rosin acid esters of polyoxyethyleneglycols) to form the STRAIN g figa g g gii culture medium. 2.5 Mls.dodecane were also. Liter (mole) sion (mole) added to the shaker flask.The flasks were gauze Covered o t lit figtqlovot Corynebaclerium sp.

, G 11-25, ATCC 21744 46 40 4t Into the mam fermentation test flastsprepared above 41 28 49 were added 5 mls. of the inoculum broth preparedas 2? g2 22 above. The flasks were then incubated at 43 sg 7 so TABLEll-Continued l, l Z-DODECANEDlOlC AClD mM millimoles We claim:

1. A process for the production of an alpha, omegaalkanedioic acid of to16 carbon atoms which comprises (l) introducing a mutated strain ofCorynebacterium capable of producing at high yield and conversion alpha,omega-alkanedioic acid of 10 to 16 carbon atoms into a composition whichhas a pH of from 6 to 9 and which comprises 1 to 50 percent by volume ofnalkane of 10 to 16 carbon atoms and from 50 to 99 percent by volume ofan aqueous culture medium comprising (a) 3 to 35 grams per liter of amember selected from the group consisting of alkali metal nitrate,calcium nitrate, magnesium nitrate, strontium nitrate, beryllium nitrateand mixtures thereof, (b) 0.3 to 10 grams per liter of alkali metalphosphate, (c) l to grams per liter of a member selected from the groupswea n fallsal nmst l. a ate a i m t magnesium acetate, strontiumacetate, beryllium acetate and mixtures thereof and (d) a nutrientsource; (2) maintaining the composition at a temperature of betweenabout 20C. and 45C. for 48 to 96 hours and (3) separating the alpha,omega-alkanedioic acid from the resulting mixture.

2. The process of claim 1 wherein the n-alkane is from 10 to 14 carbonatoms, the amount of n-alkane in the composition is 5 to 20 percent byvolume, and the amount of culture medium is to percent by volume.

3. A process for the production of an alpha, omegaalkanedioic acid of 10to 16 carbon atoms which comprises (l) introducing a mutated strain ofCorynebacterium capable of producing at high yield and conversion alpha,omega-alkanedioic acid of 10 to 16 carbon atoms into a composition whichhas a pH of from 6 to 9 and which comprises 1 to 50 percent by volume ofnalkane of 10 to 16 carbon atoms and from 50 to 99 percent by volume ofa culture medium comprising (a) 6 to 30 grams per liter of a member ofthe group consisting of sodium nitrate and potassium nitrate, (b) l to 6grams per liter of an alkali metal phosphate, (c) 2.5 to 10 grams perliter of a member selected from the group consisting of sodium acetateand potassium acetate and (d) a nutrient source; (2) maintaining thecomposition at a temperature of between about 20C. and 45C. for 48 to 96hours and (3) separating the alpha, omegaalkanedioic acid from theresulting mixture.

4. The process of claim 3 wherein the n-alkane is from 10 to 14 carbonatoms, the volume of the nalkane in the composition is 5 to 20 percentby volume, and the amount of culture medium is 80 to 95 percent byvolume.

5. The process of claim 4 wherein the n-alkane contains 12 carbon atoms.

2. The process of claim 1 wherein the n-alkane is from 10 to 14 carbonatoms, the amount of n-alkane in the composition is 5 to 20 percent byvolume, and the amount of culture medium is 80 to 95 percent by volume.3. A process for the production of an alpha, omega-alkanedioic acid of10 to 16 carbon atoms which comprises (1) introducing a mutated strainof Corynebacterium capable of producing at high yield and conversionalpha, omega-alkanedioic acid of 10 to 16 carbon atoms into acomposition which has a pH of from 6 to 9 and which comprises 1 to 50percent by volume of n-alkane of 10 to 16 carbon atoms and from 50 to 99percent by volume of a culture medium comprising (a) 6 to 30 grams perliter of a member of the group consisting of sodium nitrate andpotassium nitrate, (b) 1 to 6 grams per liter of an alkali metalphosphate, (c) 2.5 to 10 grams per liter of a member selected from thegroup consisting of sodium acetate and potassium acetate and (d) anutrient source; (2) maintaining the composition at a temperature ofbetween about 20*C. and 45*C. for 48 to 96 hours and (3) separating thealpha, omega-alkanedioic acid from the resulting mixture.
 4. The processof claim 3 wherein the n-alkane is from 10 to 14 carbon atoms, thevolume of the n-alkane in the composition is 5 to 20 percent by volume,and the amount of culture medium is 80 to 95 perCent by volume.
 5. Theprocess of claim 4 wherein the n-alkane contains 12 carbon atoms.